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Mechanisms by which electric (E) or magnetic (B) fields might be harnessed to affect tumor cell behavior remain poorly defined, presenting a barrier to translation. We hypothesized in early studies that the glycocalyx of lung cancer cells might play a role in mediating plasma membrane leak by low-frequency pulsed magnetic fields (Lf-PMF) generated on a low-energy solenoid platform. In testing glioblastoma and neuroblastoma cells known to overexpress glycoproteins rich in modifications by the anionic glycan sialic acid (Sia), exposure of brain tumor cells on the same platform to a pulse train that included a 5 min 50Hz Lf-PMF (dB/dt ∼ 2 T/s at 10 ms pulse widths) induced a very modest but significant protease leak above that of control nonexposed cells (with modest but significant reductions in long-term tumor cell viability after the 5 min exposure). Using a markedly higher dB/dt system (80 T/s pulses, 70 μs pulse-width at 5.9 cm from a MagVenture coil source) induced markedly greater leak by the same cells, and eliminating Sia by treating cells with AUS sialidase immediately preexposure abrogated the effect entirely in SH-SY5Y neuroblastoma cells, and partially in T98G glioblastoma cells. The system demonstrated significant leak (including inward leak of propidium iodide), with reduced leak at lower dB/dt in a variety of tumor cells. The ability to abrogate Lf-PMF protease leak by pretreatment with sialidase in SH-SY5Y brain tumor cells or with heparin lyase in A549 lung tumor cells indicated the importance of heavy Sia or heparan sulfate glycosaminoglycan glycocalyx modifications as dominant glycan species mediating Lf-PMF membrane leak in respective tumor cells. This "first-physical" Lf-PMF tumor glycocalyx event, with downstream cell stress, may represent a critical and "tunable" transduction mechanism that depends on characteristic anionic glycans overexpressed by distinct malignant tumors., Competing Interests: Declaration of interests None to disclose., (Published by Elsevier Inc.)

The aim of this study is to fabricate reactive oxygen species (ROS)-sensitive nanoparticles composed of succinyl β-cyclodextrin (bCDsu), memantine and thioketal linkages for application in Alzheimer's disease, and to investigate the suppression of N -methyl-d-aspartate (NMDA) receptor 1 (NMDAR1) in cells. Thioketal diamine was attached to the carboxyl group of bCDsu to produce thioketal-decorated bCDsu conjugates (bCDsu-thioketal conjugates) and memantine was conjugated with thioketal dicarboxylic acid (memantine-thioketal carboxylic acid conjugates). Memantine-thioketal carboxylic acid conjugates were attached to bCDsu-thioketal conjugates to produce bCDsu-thioketal-memantine (bCDsuMema) conjugates. SH-SY5Y neuroblastoma cells and U87MG cells were used for NMDAR1 protein expression and cellular oxidative stress. Nanoparticles of bCDsuMema conjugates were prepared by means of a dialysis procedure. Nanoparticles of bCDsuMema conjugates had small particle sizes less than 100 nm and their morphology was found to be spherical in transmission electron microscopy observations (TEM). Nanoparticles of bCDsuMema conjugates responded to H 2 O 2 and disintegrated or swelled in aqueous solution. Then, the nanoparticles rapidly released memantine according to the concentration of H 2 O 2 . In an in vivo animal imaging study, thioketal-decorated nanoparticles labelled with fluorescent dye such as chlorin e6 (Ce6) showed that the fluorescence intensity was stronger in the brain than in other organs, indicating that bCDsuMema nanoparticles can efficiently target the brain. When cells were exposed to H 2 O 2 , the viability of cells was time-dependently decreased. Memantine or bCDsuMema nanoparticles did not practically affect the viability of the cells. Furthermore, a western blot assay showed that the oxidative stress produced in cells using H 2 O 2 increased the expression of NMDAR1 protein in both SH-SY5Y and U87MG cells. Memantine or bCDsuMema nanoparticles efficiently suppressed the NMDAR1 protein, which is deeply associated with Alzheimer's disease. Fluorescence microscopy also showed that H 2 O 2 treatment induced green fluorescence intensity, which represents intracellular ROS levels. Furthermore, H 2 O 2 treatment increased the red fluorescence intensity, which represents the NMDAR1 protein, i.e., oxidative stress increases the expression of NMDAR1 protein level in both SH-SY5Y and U87MG cells. When memantine or bCDsuMema nanoparticles were treated in cells, the oxidative stress-mediated expression of NMDAR1 protein in cells was significantly decreased, indicating that bCDsuMema nanoparticles have the capacity to suppress NMDAR1 expression in brain cells, which has relevance in terms of applications in Alzheimer's disease.

High-risk neuroblastoma remains therapeutically challenging to treat, and the mechanisms promoting disease aggression are poorly understood. Here, we show that elevated expression of dihydrolipoamide S-succinyltransferase (DLST) predicts poor treatment outcome and aggressive disease in patients with neuroblastoma. DLST is an E2 component of the α-ketoglutarate (αKG) dehydrogenase complex, which governs the entry of glutamine into the tricarboxylic acid cycle (TCA) for oxidative decarboxylation. During this irreversible step, αKG is converted into succinyl-CoA, producing NADH for oxidative phosphorylation (OXPHOS). Utilizing a zebrafish model of MYCN-driven neuroblastoma, we demonstrate that even modest increases in DLST expression promote tumor aggression, while monoallelic dlst loss impedes disease initiation and progression. DLST depletion in human MYCN-amplified neuroblastoma cells minimally affected glutamine anaplerosis and did not alter TCA cycle metabolites other than αKG. However, DLST loss significantly suppressed NADH production and impaired OXPHOS, leading to growth arrest and apoptosis of neuroblastoma cells. In addition, multiple inhibitors targeting the electron transport chain, including the potent IACS-010759 that is currently in clinical testing for other cancers, efficiently reduced neuroblastoma proliferation in vitro . IACS-010759 also suppressed tumor growth in zebrafish and mouse xenograft models of high-risk neuroblastoma. Together, these results demonstrate that DLST promotes neuroblastoma aggression and unveils OXPHOS as an essential contributor to high-risk neuroblastoma. SIGNIFICANCE: These findings demonstrate a novel role for DLST in neuroblastoma aggression and identify the OXPHOS inhibitor IACS-010759 as a potential therapeutic strategy for this deadly disease., (©2021 American Association for Cancer Research.)

Neuroblastoma (NBL) is the most common extracranial childhood malignant tumor and represents a major cause of cancer-related deaths in infants. NMYC amplification or overexpression is associated with the malignant behavior of NBL tumors. In the present study, we revealed an association between long non-coding RNA (lncRNA) myocardial infarction associated transcript (MIAT) and NMYC amplification in NBL cell lines and MIAT expression in NBL tissue samples. MIAT silencing induces cell death only in cells with NMYC amplification, but in NBL cells without NMYC amplification it decreases only the proliferation. MIAT downregulation markedly reduces the NMYC expression in NMYC -amplified NBL cell lines and c-Myc expression in NMYC non-amplified NBL cell lines, but the ectopic overexpression or downregulation of NMYC did not affect the expression of MIAT. Moreover, MIAT downregulation results in decreased ornithine decarboxylase 1 (ODC1), a known transcriptional target of MYC oncogenes, and decreases the glycolytic metabolism and respiratory function. These results indicate that MIAT is an upstream regulator of NMYC and that MIAT/NMYC axis disruption induces cell death in NMYC -amplified NBL cell lines. These findings reveal a novel mechanism for the regulation of NMYC in NBL, suggesting that MIAT might be a potential therapeutic target, especially for those with NMYC amplification.

Central nervous system neuroblastoma with FOXR2 activation (CNS NB FOXR2) has recently been described as a class of brain tumors sharing common genetic events and a highly similar DNA methylation profile. Most of these tumors have previously been diagnosed as primitive neuroectodermal tumor (PNET). Whereas the entity of PNET has been removed from the WHO classification of brain tumors in its current edition, CNS neuroblastoma was kept as an entity, but still lacks any molecular detail. Here, we describe 8 cases of CNS NB FOXR2 focusing on histomorphological and immunohistochemical features and include magnetic resonance imaging (MRI) for 2 of these cases. MRI revealed large supratentorial masses in superficial location with prominent cysts and necrosis, but little edema. Diffusion and enhancement characteristics were variable. Histological analyses showed that most of the cases displayed neuronal differentiation with necrosis, endothelial proliferation, and high vascularity. Immunohistochemistry revealed strong expression of synaptophysin, MAP2, and OLIG2 as well as moderate proliferation. These findings suggest that tumors with the molecular diagnosis of CNS NB FOXR2 may fit well into the WHO entity of CNS neuroblastoma. Our findings may be helpful when establishing an integrated diagnosis and may be indispensable if molecular data are unavailable., (© 2020 American Association of Neuropathologists, Inc. All rights reserved.)

Neuroblastoma (NB) is a heterogeneous tumor affecting children. It shows a wide spectrum of clinical outcomes; therefore, development of risk stratification is critical to provide optimum treatment. Since epigenetic alterations such as DNA methylation have emerged as an important feature of both development and progression in NB, in this study, we aimed to quantify the effect of methylation of three distinct genes (RASSF1A, DCR2, and CASP8) on overall survival in NB patients. We performed a systematic review using PubMed, Embase, and Cochrane libraries. Individual patient data was retrieved from extracted Kaplan-Meier curves. Data from studies was then merged, and analysis was done on the full data set. Seven studies met the inclusion criteria. Methylation of the three genes had worse overall survival than the unmethylated arms. Five-year survival for the methylated arm of RASSF1A, DCR2, and CASP8 was 63.19% (95% CI 56.55-70.60), 57.78% (95% CI 47.63-70.08), and 56.39% (95% CI 49.53-64.19), respectively, while for the unmethylated arm, it was 93.10% (95% CI 87.40-99.1), 84.84% (95% CI 80.04-89.92), and 83.68% (95% CI 80.28-87.22), respectively. In conclusion, our results indicate that in NB patients, RASSF1A, DCR2, and CASP8 methylation is associated with poor prognosis. Large prospective studies will be necessary to confirm definitive correlation between methylation of these genes and survival taking into account all other known risk factors. (PROSPERO registration number CRD42017082264)., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2020 Waleed M. Hassan et al.)

Neuroblastoma is an important problem in children. Long noncoding RNAs (lncRNAs) exhibit important roles in tumorigenicity of neuroblastoma. However, the role and mechanism of lncRNA small nucleolar RNA host gene 16 (SNHG16) in neuroblastoma tumorigenicity remain poorly understood. Forty-six neuroblastoma samples and 28 normal tissues were harvested. The levels of SNHG16, microRNA-15b-5p (miR-15b-5p), and phosphoribosyl pyrophosphate synthetase 1 (PRPS1) were detected via quantitative reverse transcription PCR or western blot. Cell proliferation as well as cycle distribution were measured via 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide or flow cytometry. Cell metastasis was investigated via epithelial-mesenchymal transition or transwell assay. The target relationship of miR-15b-5p and SNHG16 or PRPS1 was explored via starBase and dual-luciferase reporter assay. The role of SNHG16 in neuroblastoma in vivo was analyzed using a xenograft model. We found SNHG16 and PRPS1 levels were increased in neuroblastoma tissues and cells. SNHG16 knockdown inhibited cell proliferation, increased the cell cycle distribution at G0/G1 phase, and decreased the cells at S phase. SNHG16 overexpression caused an opposite effect. SNHG16 silence suppressed neuroblastoma cell metastasis. PRPS1 knockdown constrained cell proliferation and metastasis and regulated cell cycle distribution. miR-15b-5p was sponged by SNHG16 and directly targeted PRPS1. miR-15b-5p knockdown or PRPS1 overexpression mitigated the influence of SNHG16 silence on cell cycle, proliferation, and metastasis. SNHG16 knockdown reduced xenograft tumor growth. In conclusion, SNHG16 downregulation suppressed neuroblastoma tumorigenicity by regulating cell cycle, proliferation, and metastasis via miR-15b-5p/PRPS1 axis.

Neuroblastoma resection represents a major challenge in pediatric surgery, because of the high risk of complications. Fluorescence-guided surgery (FGS) could lower this risk by facilitating discrimination of tumor from normal tissue and is gaining momentum in adult oncology. Here, we provide the first molecular-targeted fluorescent agent for FGS in pediatric oncology, by developing and preclinically evaluating a GD2-specific tracer consisting of the immunotherapeutic antibody dinutuximab-beta, recently approved for neuroblastoma treatment, conjugated to near-infrared (NIR) fluorescent dye IRDye800CW. We demonstrated specific binding of anti-GD2-IRDye800CW to human neuroblastoma cells in vitro and in vivo using xenograft mouse models. Furthermore, we defined an optimal dose of 1 nmol, an imaging time window of 4 days after administration and show that neoadjuvant treatment with anti-GD2 immunotherapy does not interfere with fluorescence imaging. Importantly, as we observed universal, yet heterogeneous expression of GD2 on neuroblastoma tissue of a wide range of patients, we implemented a xenograft model of patient-derived neuroblastoma organoids with differential GD2 expression and show that even low GD2 expressing tumors still provide an adequate real-time fluorescence signal. Hence, the imaging advancement presented in this study offers an opportunity for improving surgery and potentially survival of a broad group of children with neuroblastoma.

Therapeutic checkpoint antibodies blocking programmed death receptor 1/programmed death ligand 1 (PD-L1) signaling have radically improved clinical outcomes in cancer. However, the regulation of PD-L1 expression on tumor cells is still poorly understood. Here we show that intratumoral copper levels influence PD-L1 expression in cancer cells. Deep analysis of the The Cancer Genome Atlas database and tissue microarrays showed strong correlation between the major copper influx transporter copper transporter 1 (CTR-1) and PD-L1 expression across many cancers but not in corresponding normal tissues. Copper supplementation enhanced PD-L1 expression at mRNA and protein levels in cancer cells and RNA sequencing revealed that copper regulates key signaling pathways mediating PD-L1-driven cancer immune evasion. Conversely, copper chelators inhibited phosphorylation of STAT3 and EGFR and promoted ubiquitin-mediated degradation of PD-L1. Copper-chelating drugs also significantly increased the number of tumor-infiltrating CD8 + T and natural killer cells, slowed tumor growth, and improved mouse survival. Overall, this study reveals an important role for copper in regulating PD-L1 and suggests that anticancer immunotherapy might be enhanced by pharmacologically reducing intratumor copper levels. SIGNIFICANCE: These findings characterize the role of copper in modulating PD-L1 expression and contributing to cancer immune evasion, highlighting the potential for repurposing copper chelators as enhancers of antitumor immunity. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/19/4129/F1.large.jpg., (©2020 American Association for Cancer Research.)

Monocyte chemoattractant protein-1-induced protein 1 (MCPIP1) has a multidomain structure, which assures its pleiotropic activity. The physiological functions of this protein include repression of inflammatory processes and the prevention of immune disorders. The influence of MCPIP1 on the cell cycle of cancer cells has not been sufficiently elucidated. A previous study by our group reported that overexpression of MCPIP1 affects the cell viability, inhibits the activation of the phosphoinositide-3 kinase/mammalian target of rapamycin signalling pathway, and reduces the stability of the MYCN oncogene in neuroblastoma (NB) cells. Furthermore, a decrease in expression and phosphorylation levels of cyclin-dependent kinase (CDK) 1, which has a key role in the M phase of the cell cycle, was observed. On the basis of these previous results, the purpose of our present study was to elucidate the influence of MCPIP1 on the cell cycle of NB cells. It was confirmed that ectopic overexpression of MCPIP1 in two human NB cell lines, KELLY and BE(2)-C, inhibited cell proliferation. Furthermore, flow cytometric analyses and imaging of the cell cycle with a fluorescence ubiquitination cell-cycle indicator test, demonstrated that overexpression of MCPIP1 causes an accumulation of NB cells in the G1 phase of the cell cycle, while the possibility of an increase in G0 phase due to induction of quiescence or senescence was excluded. Additional assessment of the molecular machinery responsible for the transition between the cell-cycle phases confirmed that MCPIP1 overexpression reduced the expression of cyclins A2, B1, D1, D3, E1, and E2 and decreased the phosphorylation of CDK2 and CDK4, as well as retinoblastoma protein. In conclusion, the present results indicated a relevant impact of overexpression of MCPIP1 on the cell cycle, namely a block of the G1/S cell-cycle checkpoint, resulting in arrest of NB cells in the G1 phase., (© 2020 Wiley Periodicals, Inc.)

The purinergic P2X7 receptor (P2X7R) belongs to a family of trimeric ion channels that are gated by extracellular adenosine 5'-triphosphate (ATP). Several studies have pointed to a role of P2X7R-dependent signalling in Parkinson's disease (PD)-related neurodegeneration. The pathology of (PD) is characterized by the formation of insoluble alpha-synuclein (α-Syn) aggregates-Lewy bodies, but the mechanisms underlying α-Syn-induced dopaminergic cell death are still partially unclear. Our previous studies indicate that extracellular α-Syn directly interact with neuronal P2X7R and induces intracellular free calcium mobilization in neuronal cells. The main objective of this study was to examine the involvement of P2X7R receptor in α-Syn-induced mitochondrial dysfunction and cell death. We found that P2X7R stimulation is responsible for α-Syn-induced oxidative stress and activation of the molecular pathways of programmed cell death. Exogenous α-Syn treatment led to P2X7R-dependent decrease in mitochondrial membrane potential as well as elevation of mitochondrial ROS production resulting in breakdown of cellular energy production. Moreover, P2X7R-dependent deregulation of AMP-activated protein kinase as well as decrease in parkin protein level could be responsible for α-Syn-induced mitophagy impairment and accumulation of dysfunctional mitochondria. P2X7R might be putative pharmacological targets in molecular mechanism of extracellular α-Syn toxicity., Competing Interests: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Aims: Glioblastomas are highly aggressive brain tumors with a very poor survival rate. EEF1A2, the proto-oncogenic isoform of the EEF1A translation factor family, has been found to be overexpressed and promoting tumorigenesis in multiple cancers. Interestingly, recent studies reported reduced expression of this protein in brain tumors, drawing our attention to find the functional role and mechanism of this protein in brain tumor progression., Main Methods: Using representative cell line as models, the role of EEF1A2 in cell proliferation, migration and invasion were assessed using MTS assay, scratch wound-healing assay, transwell migration and invasion assay, respectively. Activation of key signaling pathways was assessed using western blots and real-time PCR. Finally, using immunohistochemistry we checked the protein levels of EEF1A2 in CNS tumors., Key Findings: EEF1A2 was found to increase the proliferative, migratory and invasive properties of cell lines of both glial and neuronal origin. PI3K activation directly correlated with EEF1A2 levels. Protein levels of key EMT markers viz. Twist, Snail, and Slug were increased upon ectopic EEF1A2 expression. Furthermore, EEF1A2 was found to affect the expression levels of key inflammatory cytokines, growth factors and matrix metalloproteases. IHC analysis showed that EEF1A2 is upregulated in tumor tissues compared to normal tissue., Significance: EEF1A2 acts as an oncogene in both neuronal and glial cells and triggers an EMT program via PI3K pathway. However, it shows enhanced expression in neuronal cells of the brain than the glial cells, which could explain the previously reported anomaly., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Brain tumours are among the deadliest tumours being highly resistant to currently available therapies. The proliferative behaviour of gliomas is strongly influenced by ion channel activity. Small-conductance calcium-activated potassium (SK/K Ca ) channels are a family of ion channels that are associated with cell proliferation and cell survival. A combined treatment of classical anti-cancer agents and pharmacological SK channel modulators has not been addressed yet. We used the gold-derivative auranofin to induce cancer cell death by targeting thioredoxin reductases in combination with CyPPA to activate SK channels in neuro- and glioblastoma cells. Combined treatment with auranofin and CyPPA induced massive mitochondrial damage and potentiated auranofin-induced toxicity in neuroblastoma cells in vitro. In particular, mitochondrial integrity, respiration and associated energy generation were impaired. These findings were recapitulated in patient-derived glioblastoma neurospheres yet not observed in non-cancerous HT22 cells. Taken together, integrating auranofin and SK channel openers to affect mitochondrial health was identified as a promising strategy to increase the effectiveness of anti-cancer agents and potentially overcome resistance., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. The clinical course may range from spontaneous regression towards ganglioneuroblastoma/ganglioneuroma or maturation to a very aggressive form characterized by an extensive hypoxic area. In solid tumors, extracellular microenvironment hypoxia induces the transcription of hypoxia-inducible factors (HIFs) leading to synthesis of pro-angiogenic factor, VEGF; also, it increases extracellular adenosine production from ATP breakdown. To date, the role of this nucleoside in the hypoxic/angiogenic pathway characterizing the core of cancer mass has not been investigated yet. Therefore, the aim of the present study was to analyze the adenosine effect on modulation of the HIF-1α/2α/VEGF pathway mediated through A 3 AR binding. To this end, we have used a selective A 3 AR agonist IB-MECA or antagonist VUF 5574 in an in vitro model of malignant undifferentiated and all-trans retinoic acid (RA)-differentiated SH-SY5Y cells, representing the benign form of NB. Our results have shown that specific A 3 AR stimulation induces HIF and VEGF expression through the activation of mitogen-activated protein kinase/Erk kinase signaling cascade. In conclusion, the data suggest that A 3 AR may represent a marker of NB malignancy as well as a drug target for treatment of this solid tumor. Graphical Abstract.

Background: The aim of this study was to elucidate the significance of immunohistochemical staining patterns of ALK and GD2 in peripheral neuroblastic tumors with different stages and favorable/unfavorable features. Materials and methods: 32 neuroblastomas, 7 ganglioneuroblastomas, and 1 ganglioneuroma cases were immunohistochemically stained with ALK and GD2, and the expressions were graded and correlated with differentiation, size, and favorable/unfavorable histology. Results: There was no statistically significant correlation between ALK immunopositivity and tumor differentiation or stage. Although there was no statistically significant correlation between GD2 immunopositivity and stage, the intensity and prevalence of GD2 immunostaining were statistically significantly higher in the well differentiated group and in tumors which were smaller than 10 cm. Conclusion: GD2 immunostaining levels correlated with tumor differentiation and size. ALK immunostaining was not related to tumor differentiation or stage.

Inhibitor of apoptosis proteins (IAP) are cell death regulators that bind caspases and interfere with apoptotic signalling via death receptors or intrinsic cell death pathways. BIRC4/XIAP is the most potent anti-apoptotic IAP-member and it physically interacts with caspases via its BIR2 and its BIR3 domain. These domains are also critical for the interaction with mitochondria-derived SMAC/Diablo and with the IAP protein survivin. Survivin is frequently overexpressed in neuroblastoma due to a gain of 17q and we have demonstrated that survivin confers resistance to chemotherapeutic agents and reprograms metabolism of neuroblastoma cells towards glycolysis. As regulator of mitochondrial fission and autophagy survivin acts at the crossroads of mitochondrial architecture, autophagy and cellular energy metabolism. Methods : We tested the effect of SMAC-mimetics on the XIAP/survivin axis as modulator of cellular metabolism analysing mitochondrial morphology, metabolic intermediates and cellular survival. Finally, the impact of the combined treatment was evaluated in a xenograft neuroblastoma mouse model assessing the therapy effect on tumour size and volume. Results : Here we demonstrated that XIAP sequesters significant amounts of survivin within the cell that can be mobilized by so called SMAC-mimetics. SMAC-mimetics are drugs that are designed to bind with high affinity to XIAP-BIR2 / BIR3 domains to release caspases and re-sensitize XIAP-overexpressing tumors for chemotherapy. However, SMAC-mimetic treatment releases also survivin from XIAP and thereby induces mitochondrial fragmentation, prevents ROS accumulation and leads to the Warburg effect, an unwanted side effect of this therapy. Importantly, cells that drift into a highly glycolytic state due to SMAC-mimetic treatment become also highly sensitive to non-genotoxic treatment with glycolysis inhibitors such as 2-Deoxy-D-glucose (2DG) in vitro and in vivo . Conclusion : A combinational therapy of non-genotoxic SMAC-mimetics and glycolysis-inhibitors overcomes IAP-mediated cell survival in cancer and provides therefore an attractive usage of SMAC-mimetics., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

CCAR2 (cell cycle and apoptosis regulator 2) controls a variety of cellular functions; however, its main function is to regulate cell survival and cell death in response to genotoxic and metabolic stresses. Recently, we reported that CCAR2 protects cells from apoptosis following mitochondrial stress, possibly by co-operating with Hsp60. However, it is not clear how CCAR2 and Hsp60 control cell survival and death. Here, we found that depleting CCAR2 and Hsp60 downregulated expression of survivin, a member of the inhibitor of apoptosis (IAP) family. Survivin expression in neuroblastoma tissues and human cancer cell lines correlated positively with expression of CCAR2 and Hsp60. Furthermore, high expression of CCAR2, Hsp60, and survivin was associated with poor survival of neuroblastoma patients. In summary, both CCAR2 and Hsp60 are required for expression of survivin, and both promote cancer cell survival, at least in part, by maintaining survivin expression. Therefore, CCAR2, Hsp60, and survivin are candidate tumor biomarkers and prognostic markers in neuroblastomas.

Oncogenic anaplastic lymphoma kinase (ALK) is one of the few druggable targets in neuroblastoma, and therapy resistance to ALK-targeting tyrosine kinase inhibitors (TKIs) comprises an inevitable clinical challenge. Therefore, a better understanding of the oncogenic signaling network rewiring driven by ALK is necessary to improve and guide future therapies. Here, we performed quantitative mass spectrometry-based proteomics on neuroblastoma cells treated with one of three clinically relevant ALK TKIs (crizotinib, LDK378, or lorlatinib) or an experimentally used ALK TKI (TAE684) to unravel aberrant ALK signaling pathways. Our integrated proximal proteomics (IPP) strategy included multiple signaling layers, such as the ALK interactome, phosphotyrosine interactome, phosphoproteome, and proteome. We identified the signaling adaptor protein IRS2 (insulin receptor substrate 2) as a major ALK target and an ALK TKI-sensitive signaling node in neuroblastoma cells driven by oncogenic ALK. TKI treatment decreased the recruitment of IRS2 to ALK and reduced the tyrosine phosphorylation of IRS2. Furthermore, siRNA-mediated depletion of ALK or IRS2 decreased the phosphorylation of the survival-promoting kinase Akt and of a downstream target, the transcription factor FoxO3, and reduced the viability of three ALK-driven neuroblastoma cell lines. Collectively, our IPP analysis provides insight into the proximal architecture of oncogenic ALK signaling by revealing IRS2 as an adaptor protein that links ALK to neuroblastoma cell survival through the Akt-FoxO3 signaling axis., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Chromosome 17q gains are almost invariably present in high-risk neuroblastoma cases. Here, we perform an integrative epigenomics search for dosage-sensitive transcription factors on 17q marked by H3K27ac defined super-enhancers and identify TBX2 as top candidate gene. We show that TBX2 is a constituent of the recently established core regulatory circuitry in neuroblastoma with features of a cell identity transcription factor, driving proliferation through activation of p21-DREAM repressed FOXM1 target genes. Combined MYCN/TBX2 knockdown enforces cell growth arrest suggesting that TBX2 enhances MYCN sustained activation of FOXM1 targets. Targeting transcriptional addiction by combined CDK7 and BET bromodomain inhibition shows synergistic effects on cell viability with strong repressive effects on CRC gene expression and p53 pathway response as well as several genes implicated in transcriptional regulation. In conclusion, we provide insight into the role of the TBX2 CRC gene in transcriptional dependency of neuroblastoma cells warranting clinical trials using BET and CDK7 inhibitors.

Objective: To investigate the relationship between expression of programmed cell death ligand-1(PD-L1) in the tissue of neuroblastoma (NB) and patient's clinical characteristics and prognosis. Methods: Clinical data and surgical tissue paraffin blocks of 100 newly diagnosed NB children at Sun Yat-sen University Cancer Center between January 2000 and December 2015 were collected and the expression level of PD-L1 and its' relationship with pathological parameters and survival rate were analyzed retrospectively. The ratio between groups was compared by chi-square test. Kaplan-Meier method was used for survival analysis and COX regression model was used for multivariate analysis. Results: Among 100 cases, 71 were males and 29 females; there were 5 cases of stageⅠ, 4 cases of stageⅡ, 19 cases of stage Ⅲ, 65 cases of stage Ⅳ and 7 cases of stage Ⅳs. Ten out of 62 cases (16%) were N-MYC amplified; 15 cases were in low-risk group, 18 were in medium-risk group and 67 were in high-risk group. The positive rate of PD-L1 in NB tumor tissue was 57% (57/100), of which 55 were weakly positive, 1 was moderately positive and 1 was strongly positive. The positive rates of PD-L1 expression in tumor tissues without bone metastasis were higher than those with bone metastasis(66%(39/59) vs .44%(18/41), χ(2)=4.864, P= 0.027), the positive rates of PD-L1 expression in tumor tissues pathologically diagnosed as neuroblastoma were higher than those pathologically diagnosed as ganglioneuroblastoma (61%(53/87) vs .31%(4/13), χ(2)=4.195, P= 0.041), the positive rates of PD-L1 expression in tumor tissues originated from abdominal cavity were higher than those originated from other places (61% (51/83) vs. 35%(6/17), χ(2)=3.937, P= 0.047).The 4-year event-free survival (EFS) rates of patients with PD-L1 negative and positive were 40% and 33% (χ(2)=0.009, P= 0.923), respectively. The 4-year overall survival (OS) rates of patients with PD-L1 negative and positive were 62% and 58% (χ(2)=0.294, P= 0.587). Among 33 non-high-risk patients, the 4-year EFS rates of patients with PD-L1 negative and positive were 89% and 78% (χ(2)=0.001, P= 0.965), the 4-year OS rates of patients with PD-L1 negative and positive were 100% and 96% (χ(2)=0.500, P= 0.480). Among 67 high-risk patients, the 4-year EFS rates of patients with PD-L1 negative and positive were 24% and 11% (χ(2)=1.154, P= 0.282), the 4-year OS rates of patients with PD-L1 negative and positive were 48% and 41% (χ(2)=0.692, P= 0.405). Multivariate analysis showed that N-MYC gene amplification was an independent adverse prognostic factor for OS and EFS rates of NB patients ( RR : 1.726,95 %CI :1.209-2.466; RR :1.326,95 %CI :1.014-1.736) and advanced clinical stage was an independent adverse prognostic factor for EFS rates of NB patients ( RR : 26.498, 95 %CI :3.518-199.614). Conclusions: The expression of PD-L1 in NB tumor tissues was correlated with the clinical characteristics of children. However, there were no significant differences in the prognosis of patients with or without PD-L1 expression.

The present study identified the cytotoxic effects of etomidate on the N2a neuroblastoma cell line. Etomidate induced apoptosis in N2a cells in a concentration‑dependent manner, which was confirmed by western blotting and flow cytometry. Phase contrast microscopy was used to analyze the effect of etomidate on morphological characteristics. The number of the apoptotic cells was increased and confirmed by DAPI and PI staining, which served as a characteristic hallmark of apoptosis. Additionally, etomidate led to loss of mitochondrial membrane potential and resulted in the generation of reactive oxygen species in N2a cells. The western blot analysis revealed that N2a cells treated with etomidate had a significant modulation of pro‑apoptotic proteins, includingpoly ADP‑ribose polymerase (PARP), cleaved PARP, caspase‑9 and procaspase‑3. In conclusion, the present study determined that etomidate induced cytotoxic and apoptotic effects in N2a brain tumor cells in vitro.

Background: Long non-coding RNAs (lncRNAs) are typically expressed at low levels and are inherently highly variable. This is a fundamental challenge for differential expression (DE) analysis. In this study, the performance of 25 pipelines for testing DE in RNA-seq data is comprehensively evaluated, with a particular focus on lncRNAs and low-abundance mRNAs. Fifteen performance metrics are used to evaluate DE tools and normalization methods using simulations and analyses of six diverse RNA-seq datasets., Results: Gene expression data are simulated using non-parametric procedures in such a way that realistic levels of expression and variability are preserved in the simulated data. Throughout the assessment, results for mRNA and lncRNA were tracked separately. All the pipelines exhibit inferior performance for lncRNAs compared to mRNAs across all simulated scenarios and benchmark RNA-seq datasets. The substandard performance of DE tools for lncRNAs applies also to low-abundance mRNAs. No single tool uniformly outperformed the others. Variability, number of samples, and fraction of DE genes markedly influenced DE tool performance., Conclusions: Overall, linear modeling with empirical Bayes moderation (limma) and a non-parametric approach (SAMSeq) showed good control of the false discovery rate and reasonable sensitivity. Of note, for achieving a sensitivity of at least 50%, more than 80 samples are required when studying expression levels in realistic settings such as in clinical cancer research. About half of the methods showed a substantial excess of false discoveries, making these methods unreliable for DE analysis and jeopardizing reproducible science. The detailed results of our study can be consulted through a user-friendly web application, giving guidance on selection of the optimal DE tool ( http://statapps.ugent.be/tools/AppDGE/ ).

Recent studies suggest the emerging roles of armadillo (ARM) family proteins in tumor progression. However, the functions and underlying mechanisms of ARM members in tumorigenesis and aggressiveness of neuroblastoma (NB) remain to be determined. Herein, we identify armadillo repeat containing 12 (ARMC12) as an ARM member associated with NB progression. ARMC12 promotes the growth and aggressiveness of NB cell lines. Mechanistically, ARMC12 physically interacts with retinoblastoma binding protein 4 (RBBP4) to facilitate the formation and activity of polycomb repressive complex 2, resulting in transcriptional repression of tumor suppressive genes. Blocking the interaction between ARMC12 and RBBP4 by cell-penetrating inhibitory peptide activates the downstream gene expression and suppresses the tumorigenesis and aggressiveness of NB cells. Both ARMC12 and RBBP4 are upregulated in NB tissues, and are associated with unfavorable outcome of patients. These findings suggest the crucial roles of ARMC12 in tumor progression and a potential therapeutic approach for NB.

The genetic aetiology of sporadic neuroblastoma is still largely unknown. We have identified diverse neuroblastoma susceptibility loci by genomewide association studies (GWASs); however, additional SNPs that likely contribute to neuroblastoma susceptibility prompted this investigation for identification of additional variants that are likely hidden among signals discarded by the multiple testing corrections used in the analysis of genomewide data. There is evidence suggesting the CDKN1B, coding for the cycle inhibitor p27Kip1, is involved in neuroblastoma. We thus assess whether genetic variants of CDKN1B are associated with neuroblastoma. We imputed all possible genotypes across CDKN1B locus on a discovery case series of 2101 neuroblastoma patients and 4202 genetically matched controls of European ancestry. The most significantly associated rs34330 was analysed in an independent Italian cohort of 311 cases and 709 controls. In vitro functional analysis was carried out in HEK293T and in neuroblastoma cell line SHEP-2, both transfected with pGL3-CDKN1B-CC or pGL3-CDKN1B-TT constructs. We identified an association of the rs34330 T allele (-79C/T) with the neuroblastoma risk (P combined = 0.002; OR = 1.17). The risk allele (T) of this single nucleotide polymorphism led to a lower transcription rate in cells transfected with a luciferase reporter driven by the polymorphic p27Kip1 promoter (P < 0.05). Three independent sets of neuroblastoma tumours carrying -79TT genotype showed a tendency towards lower CDKN1B mRNA levels. Our study shows that a functional variant, associated with a reduced CDKN1B gene transcription, influences neuroblastoma susceptibility., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Neuroblastoma (NB) is the most common extracranial pediatric solid tumor. Around 70% of patients with metastatic disease at diagnosis present bone-marrow infiltration, which is considered a marker of poor outcome; however, the mechanism underlying this specific tropism has to be elucidated. Tumor-derived exosomes may support metastatic progression in several tumors by interacting with the microenvironment, and may serve as tumor biomarkers. The main objective of this study is to identify an exosomal signature associated with NB metastatic bone-marrow dissemination. Therefore, the proteomic cargo of exosomes isolated from NB cell lines derived from primary tumor and bone-marrow metastasis is characterized. The comparison among exosomal proteins show 15 proteins exclusively present in primary tumor-derived exosomes, mainly involved in neuronal development, and 6 proteins in metastasis-derived exosomes related to cancer progression. Significant proteins obtain with statistical analysis performed between the two groups, reveal that primary tumor exosomes contain a higher level of proteins involved in extra-cellular matrix (ECM) assembly and adhesion, as well as in neuronal development. Exosomes isolated from bone-marrow metastasis exhibit proteins involved in ameboidal cell migration and mitochondrial activity. This work suggests that proteomic profiling of NB-derived exosomes reflects the tumor stage and may be considered as potential tumor biomarker., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nuclear lamins support the nuclear envelope and provide anchorage sites for chromatin. They are involved in DNA synthesis, transcription, and replication. It has previously been reported that the lack of Lamin A/C expression in lymphoma and leukaemia is due to CpG island promoter hypermethylation. Here, we provide evidence that Lamin A/C is silenced via this mechanism in a subset of neuroblastoma cells. Moreover, Lamin A/C expression can be restored with a demethylating agent. Importantly, Lamin A/C reintroduction reduced cell growth kinetics and impaired migration, invasion, and anchorage-independent cell growth. Cytoskeletal restructuring was also induced. In addition, the introduction of lamin Δ50, known as Progerin, caused senescence in these neuroblastoma cells. These cells were stiffer and developed a cytoskeletal structure that differed from that observed upon Lamin A/C introduction. Of relevance, short hairpin RNA Lamin A/C depletion in unmethylated neuroblastoma cells enhanced the aforementioned tumour properties. A cytoskeletal structure similar to that observed in methylated cells was induced. Furthermore, atomic force microscopy revealed that Lamin A/C knockdown decreased cellular stiffness in the lamellar region. Finally, the bioinformatic analysis of a set of methylation arrays of neuroblastoma primary tumours showed that a group of patients (around 3%) gives a methylation signal in some of the CpG sites located within the Lamin A/C promoter region analysed by bisulphite sequencing PCR. These findings highlight the importance of Lamin A/C epigenetic inactivation for a subset of neuroblastomas, leading to enhanced tumour properties and cytoskeletal changes. Additionally, these findings may have treatment implications because tumour cells lacking Lamin A/C exhibit more aggressive behaviour.

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. In this study, we examined the expression of bone morphogenetic protein receptor 2 (BMPR2) in primary NB and adjacent non-tumor samples (adrenal gland). BMPR2 expression was significantly downregulated in NB tissues, particularly in high-grade NB, and was inversely related to the expression of the NB differentiation markers ferritin and enolase. The significance of the downregulation was further explored in cultured NB cells. While enforced expression of BMPR2 decreased cell proliferation and colony-forming activity, shRNA-mediated knockdown of BMPR2 led to increased cell growth and clonogenicity. In mice, NB cells harboring BMPR2 shRNA showed significantly increased tumorigenicity compared with control cells. We also performed a retrospective analysis of NB patients and identified a significant positive correlation between tumor BMPR2 expression and overall survival. These findings suggest that BMPR2 may play an important role in the development of NB., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Purpose: To compare the pharmacokinetics of 13-cis retinoic acid (13-cisRA) between Indian and UK neuroblastoma patients receiving comparable treatment, alongside measures of toxicity and response., Methods: 13-cisRA (160 mg/m(2)/day) was administered to 36 patients ≤16 years in two divided doses. Plasma 13-cisRA concentrations were determined on days 1 and 14 of cycles 1 and 4 of treatment. Area under the plasma concentration-time curve (AUC0-6h) was estimated using non-compartment modelling. Patients were genotyped for UGT2B7, CYP3A5*3, CYP3A7*2 and *2, *3 and *4 variants of CYP2C8., Results: Marked inter-patient variability in 13-cisRA pharmacokinetics was observed. There was a trend towards a higher AUC0-6h on day 1 versus day 14 for both treatment cycles studied. Children who swallowed 13-cisRA capsules (n = 18) achieved higher AUC0-6h values compared to those who could not (n = 16) (Mean AUC 21.53 vs. 9.35 µM h, P < 0.05). Patients who were event free at 1 year tended to have higher AUC0-6h on C1D1 compared to those patients who progressed, although this did not reach significance with the number of patients studied (P = 0.08). Similarly, patients who achieved a 13-cisRA C max of ≥2 µM on C1D1 tended to have higher median EFS compared to those who did not (17.0 vs. 8.1 months). UGT2B7, CYP2C8*2/*3/*4 or CYP3A5*3 genotype did not have any effect on 13-cisRA pharmacokinetics., Conclusions: Method of administration markedly affects 13-cisRA pharmacokinetics in Indian neuroblastoma patients, supporting similar findings in UK patients. An appropriate oral liquid formulation of 13-cisRA that can be administered to all children with neuroblastoma is urgently needed on an international level.

As high-risk neuroblastoma (NB) has a poor prognosis, new therapeutic modalities are needed. We therefore investigated the susceptibility of NB cells to γ-secretase inhibitor I (GSI-I). NOTCH signaling activity, the cellular effects of GSI-I and its mechanisms of cytotoxicity were evaluated in NB cells in vitro and in vivo. The results show that NOTCH signaling is relevant for human NB cells. Of the GSIs screened in vitro GSI-I was the most effective inhibitor of NB cells. Both MYCN-amplified and non-amplified NB cells were susceptible to GSI-I. Among the targets of GSI-I in NB cells were NOTCH and the proteasome. GSI-I caused G2/M arrest that was enhanced by acute activation of MYCN and led to mitotic dysfunction. GSI-I also induced proapoptotic NOXA. Survival of mice bearing an MYCN non-amplified orthotopic patient-derived NB xenograft was significantly prolonged by systemic GSI-I, associated with mitotic catastrophe and reduced angiogenesis, and without evidence of intestinal toxicity. In conclusion, the activity of GSI-I on multiple targets in NB cells and the lack of gastrointestinal toxicity in mice are advantageous and merit further investigations of GSI-I in NB.

Amplification or overexpression of MYCN is associated with poor prognosis of human neuroblastoma. We have recently defined a MYCN-dependent transcriptional signature, including protein arginine methyltransferase 1 (PRMT1), which identifies a subgroup of patients with high-risk disease. Here we provide several lines of evidence demonstrating PRMT1 as a novel regulator of MYCN and implicating PRMT1 as a potential therapeutic target in neuroblastoma pathogenesis. First, we observed a strong correlation between MYCN and PRMT1 protein levels in primary neuroblastoma tumors. Second, MYCN physically associates with PRMT1 by direct protein-protein interaction. Third, depletion of PRMT1 through siRNA knockdown reduced neuroblastoma cell viability and MYCN expression. Fourth, we showed that PRMT1 regulates MYCN stability and identified MYCN as a novel substrate of PRMT1. Finally, we demonstrated that mutation of putatively methylated arginine R65 to alanine decreased MYCN stability by altering phosphorylation at residues serine 62 and threonine 58. These results provide mechanistic insights into the modulation of MYCN oncoprotein by PRMT1, and suggest that targeting PRMT1 may have a therapeutic impact on MYCN-driven oncogenesis.

Background: Although survival for neuroblastoma patients has dramatically improved in recent years, a substantial number of children in the high-risk subgroup still die., Methods: We aimed to define a subgroup of ultra-high-risk patients from within the high-risk cohort. We used advanced morphometric approaches to quantify and characterise blood vessels, reticulin fibre networks, collagen type I bundles, elastic fibres and glycosaminoglycans in 102 high-risk neuroblastomas specimens. The Kaplan-Meier method was used to correlate the analysed elements with survival., Results: The organisation of blood vessels and reticulin fibres in neuroblastic tumours defined an ultra-high-risk patient subgroup with 5-year survival rate <15%. Specifically, tumours with irregularly shaped blood vessels, large sinusoid-like vessels, smaller and tortuous venules and arterioles and with large areas of reticulin fibres forming large, crosslinking, branching and haphazardly arranged networks were linked to the ultra-high-risk phenotype., Conclusions: We demonstrate that quantification of tumour stroma components by morphometric techniques has the potential to improve risk stratification of neuroblastoma patients.

Background: Neuroblastoma, a tumor of the developing sympathetic nervous system, is a common childhood neoplasm that is often lethal. Mitochondrial DNA (mtDNA) mutations have been found in most tumors including neuroblastoma. We extracted mtDNA data from a cohort of neuroblastoma samples that had undergone Whole Exome Sequencing (WES) and also used snap-frozen samples in which mtDNA was entirely sequenced by Sanger technology. We next undertook the challenge of determining those mutations that are relevant to, or arisen during tumor development. The bioinformatics pipeline used to extract mitochondrial variants from matched tumor/blood samples was enriched by a set of filters inclusive of heteroplasmic fraction, nucleotide variability, and in silico prediction of pathogenicity., Results: Our in silico multistep workflow applied both on WES and Sanger-sequenced neuroblastoma samples, allowed us to identify a limited burden of somatic and germline mitochondrial mutations with a potential pathogenic impact., Conclusions: The few singleton germline and somatic mitochondrial mutations emerged, according to our in silico analysis, do not appear to impact on the development of neuroblastoma. Our findings are consistent with the hypothesis that most mitochondrial somatic mutations can be considered as 'passengers' and consequently have no discernible effect in this type of cancer., Competing Interests: The authors declare no conflicts of interest.

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood. Our previous studies have shown that hypoxia-inducible factor 2 alpha (HIF-2α), one member of the bHLH-PAS transcription factor family, facilitates the progression of NB under non-hypoxic conditions. However, the mechanisms underlying HIF-2α expression in NB still remain largely unknown. Herein, through analyzing the computational algorithm programs, we identified microRNA-558 (miR-558) as a crucial regulator of HIF-2α expression in NB. We demonstrated that miR-558 promoted the expression of HIF-2α at translational levels in NB cells through recruiting Argonaute 2 (AGO2). Mechanistically, miR-558 directly bound with its complementary site within 5'-untranslated region (5'-UTR) to facilitate the binding of AGO2 to eukaryotic translation initiation factor 4E (eIF4E) binding protein 1, resulting in increased eIF4E enrichment and HIF-2α translation. In addition, miR-558 promoted the growth, invasion, metastasis, and angiogenesis of NB cells in vitro and in vivo, and these biological features were rescued by knockdown of AGO2, eIF4E, or HIF-2α. In clinical NB specimens, miR-558, AGO2, and eIF4E were highly expressed and positively correlated with HIF-2α expression. Patients with high miR-558, HIF-2α, AGO2, or eIF4E levels had lower survival probability. Taken together, these results demonstrate that miR-558 facilitates the expression of HIF-2α through bindingto its 5'-UTR, thus promoting the tumorigenesis and aggressiveness of NB., Competing Interests: The authors declare no conflict of interest.

Aims: We investigated the neuroprotective effects and action mechanism of three major compounds [daucosterol (Dau), pectolinarin (Pec), and astragalin (Ast)] isolated from edible plants against H2O2-induced cell death of human brain neuroblastoma SK-N-SH cells., Main Methods: Cytotoxicity was determined by MTT and lactate dehydrogenase (LDH) assays. Apoptotic cell death was monitored by annexin V-FITC/PI double staining and by TUNEL assay. The formation of reactive oxygen species (ROS), expression of antioxidant enzymes and phosphorylation of mitogen-activated protein kinase (MAPK) were determined by 2,7-dichlorofluorescein diacetate (DCF-DA) assay, RT-PCR, and western blotting, respectively., Key Findings: The ethyl acetate fractions from Cirsium setidens (CSEA) and Aster scaber (ASEA) showed neuroprotective effects in SK-N-SH cells. The phytochemicals were isolated from CSEA and ASEA and identified by spectral analyses, as β-sitosterol, Dau, Pec, Ast, or isoquercitrin. Pretreatment with Dau, Pec, or Ast showed protective effects against H2O2-induced cell death and inhibited ROS generation by oxidative stress. HO-1 mRNA and protein levels were increased by the presence of H2O2 and were further elevated by pretreatment with Dau and Ast. Dau pretreatment resulted in further increases of H2O2-induced enhancement in levels of CAT and SOD2. Pretreatment with Dau, Pec, and Ast inhibited phosphorylation of MAPK, such as extracellular protein regulated protein kinase, p38, and c-Jun N-terminal kinase by H2O2., Significance: Dau exerts its neuroprotective effects by down regulation of MAPK pathways and upregulation of the HO-1, CAT and SOD2 antioxidant genes and is associated with reduced oxidative stress in SK-N-SH cells., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Tissue homeostasis requires accurate control of cell proliferation, differentiation and chromosome segregation. Drosophila sas-4 and aurA mutants present brain tumours with extra neuroblasts (NBs), defective mitotic spindle assembly and delayed mitosis due to activation of the spindle assembly checkpoint (SAC). Here we inactivate the SAC in aurA and sas-4 mutants to determine whether the generation of aneuploidy compromises NB proliferation. Inactivation of the SAC in the sas-4 mutant impairs NB proliferation and disrupts euploidy. By contrast, disrupting the SAC in the aurA mutant does not prevent NB amplification, tumour formation or chromosome segregation. The monitoring of Mad2 and cyclin B dynamics in live aurA NBs reveals that SAC satisfaction is not coupled to cyclin B degradation. Thus, the NBs of aurA mutants present delayed mitosis, with accurate chromosome segregation occurring in a SAC-independent manner. We report here the existence of an Aurora A-dependent mechanism promoting efficient, timed cyclin B degradation.

Aim: To define the DNA methylation landscape of neuroblastoma and its clinicopathological impact., Materials & Methods: Microarray DNA methylation data were analyzed and associated with functional/regulatory genome annotation data, transcriptional profiles and clinicobiological parameters., Results: DNA methylation changes in neuroblastoma affect not only promoters but also intragenic and intergenic regions at cytosine-phosphate-guanine (CpG) and non-CpG sites, and target functional chromatin domains of development and cancer-related genes such as CCND1. Tumors with diverse clinical risk showed differences affecting CpG and, remarkably, non-CpG sites. Non-CpG methylation observed essentially in clinically favorable cases was associated with the differentiation status of neuroblastoma and expression of key genes such as ALK., Conclusion: This epigenetic fingerprint of neuroblastoma provides new insights into the pathogenesis and clinical behavior of this pediatric tumor.

Despite multimodality treatment, the long-term survival of high-risk patients with neuroblastomas is below 50%. New anti-mitotic drugs against targets, such as polo-like kinase 1 (PLK1), are being evaluated in early phase clinical trials. PLK1 phosphorylates the translationally controlled tumor protein (TCTP). We investigated the expression of PLK1 and the phosphorylated substrate, pTCTP, by immunostaining eighty-eight neuroblastomas. Digitally scanned slides were scored using image analysis software. The median PLK1 and pTCTP proliferation indices (PIs) were 4.6 and 1% respectively. There was moderate positive correlation between PLK1 and pTCTP (ρ = 0.65). The PIs for both markers were significantly higher in neuroblastomas from patients with adverse clinical (advanced-stage, high-risk group, primary abdominal compared to extra-abdominal sites), biological (MYCN amplification, 1p deletion, 17q gain) and pathological (undifferentiated or poorly differentiated status, high mitosis-karyorrhexis index, [MKI], unfavorable histology) factors. Using Cox regression models, higher-than-median PLK1 and pTCTP PIs were associated with a shorter overall survival (OS) and event-free survival (EFS) in the univariate analyses. In the multivariate analyses, a high PLK1 PI count was associated with significantly shorter OS and EFS, independent of MYCN amplification and MKI; in addition, the significantly shorter EFS was independent of the risk-group. After adjustment for MKI and MYCN amplification, and for risk-group, high pTCTP PI was also associated with significantly shorter OS. Our study shows that PLK1 provides valuable prognostic information in patients with neuroblastomas.

Neuroblastoma (NB) is the most common solid extracranial tumor in children. However, the molecular mechanism of NB remains to be elucidated. In the present study, reverse transcription quantitative polymerase chain reaction data demonstrated that the expression of Sam68 was significantly upregulated in NB tissues compared with their matched adjacent normal tissues. Furthermore, it was revealed that reduced expression of miR‑203 and increased expression of Sam68 co‑existed in NB tissues. Knockdown of Sam68 reduced the proliferation, migration and invasion of human SK‑N‑SH and SH‑SY5Y NB cells. Similarly, overexpression of miR‑203 also inhibited the proliferation, migration and invasion of these two cell lines. It was further demonstrated that the protein expression level of Sam68 was negatively mediated by miR‑203 in the SK‑N‑SH and SH‑SY5Y NB cells. Additionally, data from a dual luciferase reporter assay confirmed that miR‑203 directly targeted Sam68 by binding to its 3'‑untranslated region. In conclusion, the present study suggested for the first time, to the best of our knowledge, that the aberrant downregulation of miR‑203 may contribute to the aberrant upregulation of Sam68 in NB and that miR‑203 has an inhibitory role in malignant progression of NB by targeting Sam68. The present study provided evidence to support miR-203/Sam68 as a novel diagnostic or therapeutic targets for NB.

Among the primary brain tumors, glioblastoma multiforme (GBM) has a radical proliferation ability that complicates the therapeutic modulation of cancer progression. The majority of GBM patients have a low survival rate (<1 year) due to radical tumor growth and late cancer diagnosis. Previous reports have shown that astrocytes have a specific metabolic organization that includes the production of lactate, the storage of glycogen, and use of lactate to support neurons which possess higher capacity of metabolism compared to neurons. We hypothesized that these characteristics of astrocytes could contribute to enhanced proliferation of GBM compared to neuroblastoma (NB). Here, we show that U87MG cells (a model of GBM) proliferate more rapidly than SH-SY5Y cells (a model of NB). A higher extracellular acidification rate and maximal mitochondrial oxygen consumption rate were observed in U87MG cells compared to SH-SY5Y cells. The expression levels of lactate dehydrogenase (LDH)-A and LDH-B were higher in U87MG cells and primary cultured astrocytes than in SH-SY5Y cells and neurons. Furthermore, the mRNA levels of succinate dehydrogenase and peroxisome proliferator-activated receptor-γ were high in U87MG cells, suggesting that these cells have high capacity for mitochondrial metabolism and uptake of fatty acids related to synthesis of the cell membrane, respectively. Taken together, we demonstrate that GBM cells are characterized by activation of the LDH-expression-related glycolytic pathway and mitochondrial metabolic capacity, suggesting two innate properties of astrocytes that could provide a driving force for the growth ability of GBM. Based on these findings, we propose that therapeutic approaches aimed at treating GBM could target LDH for modulating the metabolic properties of GBM cells.

Neuroblastoma is a predominantly p53 wild-type (wt) tumour and MDM2-p53 antagonists offer a novel therapeutic strategy for neuroblastoma patients. RG7388 (Roche) is currently undergoing early phase clinical evaluation in adults. This study assessed the efficacy of RG7388 as a single-agent and in combination with chemotherapies currently used to treat neuroblastoma in a panel of neuroblastoma cell lines. RG7388 GI50 concentrations were determined in 21 p53-wt and mutant neuroblastoma cell lines of varying MYCN, MDM2 and p14(ARF) status, together with MYCN-regulatable Tet21N cells. The primary determinant of response was the presence of wt p53, and overall there was a >200-fold difference in RG7388 GI50 concentrations for p53-wt versus mutant cell lines. Tet21N MYCN+ cells were significantly more sensitive to RG7388 compared with MYCN- cells. Using median-effect analysis in 5 p53-wt neuroblastoma cell lines, selected combinations of RG7388 with cisplatin, doxorubicin, topotecan, temozolomide and busulfan were synergistic. Furthermore, combination treatments led to increased apoptosis, as evident by higher caspase-3/7 activity compared to either agent alone. These data show that RG7388 is highly potent against p53-wt neuroblastoma cells, and strongly supports its further evaluation as a novel therapy for patients with high-risk neuroblastoma and wt p53 to potentially improve survival and/or reduce toxicity.

The immunosurveillance mechanisms governing high-risk neuroblastoma (HR-NB), a major pediatric malignancy, have been elusive. We identify a potential role for natural killer (NK) cells, in particular the interaction between the NK receptor NKp30 and its ligand, B7-H6, in the metastatic progression and survival of HR-NB after myeloablative multimodal chemotherapy and stem cell transplantation. NB cells expressing the NKp30 ligand B7-H6 stimulated NK cells in an NKp30-dependent manner. Serum concentration of soluble B7-H6 correlated with the down-regulation of NKp30, bone marrow metastases, and chemoresistance, and soluble B7-H6 contained in the serum of HR-NB patients inhibited NK cell functions in vitro. The expression of distinct NKp30 isoforms affecting the polarization of NK cell functions correlated with 10-year event-free survival in three independent cohorts of HR-NB in remission from metastases after induction chemotherapy (n = 196, P < 0.001), adding prognostic value to known risk factors such as N-Myc amplification and age >18 months. We conclude that the interaction between NKp30 and B7-H6 may contribute to the fate of NB patients and that both the expression of NKp30 isoforms on circulating NK cells and the concentration of soluble B7-H6 in the serum may be clinically useful as biomarkers for risk stratification., (Copyright © 2015, American Association for the Advancement of Science.)

Approximately half of poor prognosis neuroblastomas (NBs) are characterized by pathognomonic MYCN gene amplification and MYCN over-expression. Here we present data showing that short-interfering RNA mediated depletion of the protein arginine methyltransferase 5 (PRMT5) in cell-lines representative of NBs with MYCN gene amplification leads to greatly impaired growth and apoptosis. Growth suppression is not apparent in the MYCN-negative SH-SY5Y NB cell-line, or in two immortalized human fibroblast cell-lines. Immunoblotting of NB cell-lines shows that high PRMT5 expression is strongly associated with MYCN-amplification (P < 0.004, Mann-Whitney U-test) and immunohistochemical analysis of primary NBs reveals that whilst PRMT5 protein is ubiquitously expressed in the cytoplasm of most cells, MYCN-amplified tumours exhibit pronounced nuclear PRMT5 staining. PRMT5 knockdown in MYCN-overexpressing cells, including the SHEP-21N cell-line with inducible MYCN expression leads to a dramatic decrease in MYCN protein and MYCN-associated cell-death in SHEP-21N cells. Quantitative gene expression analysis and cycloheximide chase experiments suggest that PRMT5 regulates MYCN at a post-transcriptional level. Reciprocal co-immunoprecipitation experiments demonstrated that endogenous PRMT5 and MYCN interact in both SK-N-BE(2)C and NGP cell lines. By using liquid chromatography - tandem mass spectrometry (LC-MS/MS) analysis of immunoprecipitated MYCN protein, we identified several potential sites of arginine dimethylation on the MYCN protein. Together our studies implicate PRMT5 in a novel mode of MYCN post-translational regulation and suggest PRMT5 plays a major role in NB tumorigenesis. Small-molecule inhibitors of PRMT5 may therefore represent a novel therapeutic strategy for neuroblastoma and other cancers driven by the MYCN oncogene., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Neuroblastoma inflicts mostly on children, and the pathogenesis remains elusive. Clinical diagnosis and therapeutic approaches are still on the incipient stage, so further understanding of the molecular and cellular mechanisms of the disease is necessary. Inflammation has been commonly regarded as a hallmark in tumorigenesis and development, and we identified a new inflammatory factor, HMGB1, is considerably increased in neuroblastoma. Our study shows that HMGB1 induces autophagy in Schwann cells through activation of TLR4, and knockdown of TLR4 obviates the HMGB1-induced autophagy. The HMGB1-induced autophagy is through classical pathway, as deficiency of Beclin 1 deprived autophagy in Schwann cells. Coculture of neuroblastoma with Schwann cells pretreated with HMGB1 promoted the proliferation of neuroblastoma cells, and if Beclin 1 is knocked down in Schwann cells, no promotion effects is observed. Taken together, our study demonstrates that HMGB1-induced autophagy in Schwann cells contributes to neuroblastoma cell proliferation, thus providing a potential therapeutic approach on neuroblastoma development.

LIN28 has emerged as an oncogenic driver in a number of cancers, including neuroblastoma (NB). Overexpression of LIN28 correlates with poor outcome in NB, therefore drugs that impact the LIN28/Let-7 pathway could be beneficial in treating NB patients. The LIN28/Let-7 pathway affects many cellular processes including the regulation of cancer stem cells and glycolytic metabolism. Polyamines, regulated by ornithine decarboxylase (ODC) modulate eIF-5A which is a direct regulator of the LIN28/Let-7 axis. We propose that therapy inhibiting ODC will restore balance to the LIN28/Let-7 axis, suppress glycolytic metabolism, and decrease MYCN protein expression in NB. Difluoromethylornithine (DFMO) is an inhibitor of ODC in clinical trials for children with NB. In vitro experiments using NB cell lines, BE(2)-C, SMS-KCNR, and CHLA90 show that DFMO treatment reduced LIN28B and MYCN protein levels and increased Let-7 miRNA and decreased neurosphere formation. Glycolytic metabolic activity decreased with DFMO treatment in vivo. Additionally, sensitivity to DFMO treatment correlated with LIN28B overexpression (BE(2)-C>SMS-KCNR>CHLA90). This is the first study to demonstrate that DFMO treatment restores balance to the LIN28/Let-7 axis and inhibits glycolytic metabolism and neurosphere formation in NB and that PET scans may be a meaningful imaging tool to evaluate the therapeutic effects of DFMO treatment.

Background: Accumulating evidence implicates the transcription factor NF-κB as a positive mediator of tumor metastasis, but the molecular mechanism(s) involved in this process remains largely unknown. In this study, we investigated the role of NF-κB signaling pathway in the regulation of CXC chemokine receptor-4 (CXCR4) in neuroblastoma metastasis., Material and Methods: NF-κB, CXCR4 mRNA and protein expression were measured by RT-PCR, and Western blot. Tumor necrosis factor-α (TNF-α) was used to induce the upregulation of NF-κB and CXCR4. The knockdown of NF-κB and CXCR4 was achieved by PDTC. Transwell assay was used to investigate the role of NF-κB (P65) in neuroblastoma cell migration and invasion. An in vitro co-culture system was established to investigate the role of tumor microenvironment in regulation of the NF-κB signaling pathway., Results: Over-expression of NF-κB (p65) promoted tumor migration and invasion through the upregulation of CXCR4; however, knockdown of NF-κB(P65) inhibited tumor migration and invasion through blocking the expression of CXCR4. Consistently, in the co-culture system, the expression of CXCR4 was partly dependent on the expression of NF-κB (p65)., Conclusions: Our studies reveal critical roles for the NF-κB signaling pathway in neuroblastoma migration and invasion. The mechanism may be through up-regulation of CXCR4, mediated by the NF-κB signaling pathways. Targeting NF-κB signalling pathways and ultimately CXCR4 could be a strategy in neuroblastoma therapy.

Background: FOXO transcription factors control cellular levels of reactive oxygen species (ROS) which critically contribute to cell survival and cell death in neuroblastoma. In the present study we investigated the regulation of C10orf10/DEPP by the transcription factor FOXO3. As a physiological function of C10orf10/DEPP has not been described so far we analyzed its effects on cellular ROS detoxification and death sensitization in human neuroblastoma cells., Methods: The effect of DEPP on cellular ROS was measured by catalase activity assay and live cell fluorescence microscopy using the ROS-sensitive dye reduced MitoTracker Red CM-H2XROS. The cellular localization of DEPP was determined by confocal microscopy of EYFP-tagged DEPP, fluorescent peroxisomal- and mitochondrial probes and co-immunoprecipitation of the PEX7 receptor., Results: We report for the first time that DEPP regulates ROS detoxification and localizes to peroxisomes and mitochondria in neuroblastoma cells. FOXO3-mediated apoptosis involves a biphasic ROS accumulation. Knockdown of DEPP prevented the primary and secondary ROS wave during FOXO3 activation and attenuated FOXO3- and H2O2-induced apoptosis. Conditional overexpression of DEPP elevates cellular ROS levels and sensitizes to H2O2 and etoposide-induced cell death. In neuronal cells, cellular ROS are mainly detoxified in peroxisomes by the enzyme CAT/catalase. As DEPP contains a peroxisomal-targeting-signal-type-2 (PTS2) sequence at its N-terminus that allows binding to the PEX7 receptor and import into peroxisomes, we analyzed the effect of DEPP on cellular detoxification by measuring enzyme activity of catalase. Catalase activity was reduced in DEPP-overexpressing cells and significantly increased in DEPP-knockdown cells. DEPP directly interacts with the PEX7 receptor and localizes to the peroxisomal compartment. In parallel, the expression of the transcription factor peroxisome proliferator-activated receptor gamma (PPARG), a critical regulator of catalase enzyme activity, was strongly upregulated in DEPP-knockdown cells., Conclusion: The combined data indicate that in neuroblastoma DEPP localizes to peroxisomes and mitochondria and impairs cellular ROS detoxification, which sensitizes tumor cells to ROS-induced cell death.

OSU-03012, a 3-phosphoinositide-dependent kinase-1 (PDK1) inhibitor, destabilizes MYCN and MYC proteins in neuroblastoma cells. However, AKT phosphorylation is barely detectable in neuroblastoma cells under normal culture conditions whether treated with OSU-03012 or not. This observation suggests that PDK1 is not the main target of OSU-03012 to destabilize MYC and MYCN in neuroblastoma cells. In the present study, we explored one of the possible mechanisms by which OSU-03012 destabilizes MYC and MYCN. Since Aurora kinase A is reported to phosphorylate GSK3β, leading to its inactivation, we hypothesized that one of the targets of OSU-03012 is Aurora kinase A. Comparative analysis of OSU-03012 and VX-680, a potent and specific inhibitor of Aurora kinases, showed that both inhibitors destabilized MYC and MYCN and were significantly growth suppressive to neuroblastoma cell lines. In silico molecular docking analysis further showed that the calculated interaction energy between Aurora kinase A and OSU-03012 was -109.901 kcal/mol, which was lower than that (-89.273 kcal/mol) between Aurora kinase A and FXG, an Aurora kinase-specific inhibitor. Finally, an in vitro Aurora kinase A inhibition assay using a recombinant Aurora kinase A showed that OSU-03012 significantly inhibited Aurora kinase A, although it was weaker in potency than that of VX-680. Thus, OSU-03012 has a likelihood of binding to and inhibiting Aurora kinase A in vivo. These results suggest that OSU-03012 affects multiple cellular targets, including Aurora kinase A, to exhibit its growth suppressive and MYC and MYCN-destabilizing effects on neuroblastoma and other cancer cells.

Background: Neuroblastoma (NB) is the most common extracranial solid tumor in children. NB tumors and derived cell lines are phenotypically heterogeneous. Cell lines are classified by phenotype, each having distinct differentiation and tumorigenic properties. The neuroblastic phenotype is tumorigenic, has neuronal features and includes stem cells (I-cells) and neuronal cells (N-cells). The non-neuronal phenotype (S-cell) comprises cells that are non-tumorigenic with features of glial/smooth muscle precursor cells. This study identified miRNAs associated with each distinct cell phenotypes and investigated their role in regulating associated differentiation and tumorigenic properties., Methods: A miRNA microarray was performed on the three cell phenotypes and expression verified by qRT-PCR. miRNAs specific for certain cell phenotypes were modulated using miRNA inhibitors or stable transfection. Neuronal differentiation was induced by RA; non-neuronal differentiation by BrdU. Changes in tumorigenicity were assayed by soft agar colony forming ability. N-myc binding to miR-375 promoter was assayed by chromatin-immunoprecipitation., Results: Unsupervised hierarchical clustering of miRNA microarray data segregated neuroblastic and non-neuronal cell lines and showed that specific miRNAs define each phenotype. qRT-PCR validation confirmed that increased levels of miR-21, miR-221 and miR-335 are associated with the non-neuronal phenotype, whereas increased levels of miR-124 and miR-375 are exclusive to neuroblastic cells. Downregulation of miR-335 in non-neuronal cells modulates expression levels of HAND1 and JAG1, known modulators of neuronal differentiation. Overexpression of miR-124 in stem cells induces terminal neuronal differentiation with reduced malignancy. Expression of miR-375 is exclusive for N-myc-expressing neuroblastic cells and is regulated by N-myc. Moreover, miR-375 downregulates expression of the neuronal-specific RNA binding protein HuD., Conclusions: Thus, miRNAs define distinct NB cell phenotypes. Increased levels of miR-21, miR-221 and miR-335 characterize the non-neuronal, non-malignant phenotype and miR-335 maintains the non-neuronal features possibly by blocking neuronal differentiation. miR-124 induces terminal neuronal differentiation with reduction in malignancy. Data suggest N-myc inhibits neuronal differentiation of neuroblastic cells possibly by upregulating miR-375 which, in turn, suppresses HuD. As tumor differentiation state is highly predictive of patient survival, the involvement of these miRNAs with NB differentiation and tumorigenic state could be exploited in the development of novel therapeutic strategies for this enigmatic childhood cancer.

Cucurbitacin E (CuE) is a natural compound previously shown to have anti-feedant, antioxidant and antitumor activities as well as a potent chemo-preventive action against cancer. The present study investigates its anti-proliferative property using MTT assay; CuE demonstrated cytotoxic activity against malignant glioma GBM 8401 cells and induced cell cycle G2/M arrest in these cells. CuE-treated cells accumulated in metaphase (CuE 2.5-10 μM) as determined using MPM-2 by flow cytometry. We attempted to characterize the molecular pathways responsible for cytotoxic effects of CuE in GBM 8401 cells. We studied the genome-wide gene expression profile on microarrays and molecular networks by using pathway analysis tools of bioinformatics. The CuE reduced the expression of 558 genes and elevated the levels of 1354 genes, suggesting an existence of the common pathways involved in induction of G2/M arrest. We identified the RB (GADD45β and GADD45γ) and the p53 (GADD45α) signaling pathways as the common pathways, serving as key molecules that regulate cell cycle. Results indicate that CuE produced G2/M arrest as well as the upregulation of GADD45 γ and binding with CDC2. Both effects increased proportionally with the dose of CuE, suggesting that the CuE-induced mitosis delay is regulated by GADD45γ overexpression. Our findings suggest that, in addition to the known effects on cancer prevention, CuE may have antitumor activity in glioma therapy.

Despite intensive research efforts and therapeutic advances over the last few decades, the pediatric neural crest tumor, neuroblastoma, continues to be responsible for over 15% of pediatric cancer deaths. Novel therapeutic options are needed for this tumor. Recently, investigators have shown that mice with syngeneic murine gliomas treated with an engineered, neuroattenuated oncolytic herpes simplex virus-1 (oHSV), M002, had a significant increase in survival. M002 has deletions in both copies of the γ 1 34.5 gene, enabling replication in tumor cells but precluding infection of normal neural cells. We hypothesized that M002 would also be effective in the neural crest tumor, neuroblastoma. We showed that M002 infected, replicated, and decreased survival in neuroblastoma cell lines. In addition, we showed that in murine xenografts, treatment with M002 significantly decreased tumor growth, and that this effect was augmented with the addition of ionizing radiation. Importantly, survival could be increased by subsequent doses of radiation without re-dosing of the virus. Finally, these studies showed that the primary entry protein for oHSV, CD111 was expressed by numerous neuroblastoma cell lines and was also present in human neuroblastoma specimens. We concluded that M002 effectively targeted neuroblastoma and that this oHSV may have potential for use in children with unresponsive or relapsed neuroblastoma.